Extrachromosomal circular DNA in cancer: history, current knowledge, and methods. Discoveries of extrachromosomal circles of DNA in normal and tumor cells.
eccDNAs are apoptotic products with high innate immunostimulatory activity. Molecular size and circularity of DNA in cells of mammals and higher plants. For a well-trained molecular biologist, it takes ~1–2 d to purify eccDNAs, another 5–6 d to carry out Nanopore library preparation and sequencing, and 1–5 d for an experienced bioinformatic scientist to analyze the data. Collectively, our protocol will facilitate eccDNA identification and characterization, and has the potential for diagnostic and clinical applications. Accordingly, we developed a full-length eccDNA caller (Flec) to call the consensus sequence of multiple tandem copies of eccDNA contained within the debranched rolling-circle amplification product and map the consensus to its genomic origin. Additionally, we developed a full-length eccDNA sequencing technique by combining rolling-circle amplification with Nanopore sequencing. Here we describe a new three-step eccDNA purification (3SEP) procedure that adds a step to recover circular DNA, but not linear DNA that escape from the exonuclease digestion, whereby 3SEP results in eccDNA preparations with high purity and reproducibility. Owing to eccDNA’s low abundance and heterogeneous size, the current purification methods are not efficient in obtaining pure eccDNA. The current eccDNA purification methods mainly rely on depleting linear DNAs by exonuclease digestion after obtaining crude circles by alkaline lysis.
One hurdle that has prevented our understanding of eccDNA is the difficulty in obtaining pure eccDNA from cells. However, its biogenesis and function have just begun to be elucidated. Extrachromosomal circular DNA (eccDNA) was discovered more than half a century ago.
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